EDIBLE FAT-CONTINUOUS EMULSION COMPRISING 1 -DEOXYNOJIRIMYCIN AND LECITHIN

Field of the Invention

The present invention relates to an edible fat- or oil-continuous emulsion, which edible emulsion comprises a continuous fat or oil phase, a dispersed water phase, the emulsion further comprising 1 -deoxynojirimycin (DNJ) and lecithin, wherein the lecithin comprises phosphatidylcholine (PC), phosphatidylinositol (PI), and

phosphatidylethanolamine (PE) wherein the weight ratio (PC+PI)/PE in the emulsions is at least 2.5.

The invention further relates to a process for preparing such emulsion, as well as the use of such emulsion, wherein the emulsion is combined with edible matter comprising carbohydrates, in such an amount that the resulting foodstuff contains from 0.001 to 0.03 wt % of DNJ on carbohydrate basis.

Background of the invention

Many consumers in western society are becoming increasingly health-conscious, and in particular regarding their diet. This can relate to amount of intake, calorie intake, origin of food (e.g. organic), level of processing, but also real or perceived health benefits. An area of concern, both for individuals as well as for policymakers and industrial food producers, is the blood glucose concentration after consuming carbohydrate-rich foods. Such carbohydrate-rich foods may, in some instances (e.g. when these concern mainly sugar and/or high-glycaemic carbohydrates, in particular in the absence of fiber, protein and/or fat) lead to high glucose levels in blood some time (e.g. 10-60 minutes) after consuming them. Such high levels of post prandial (i.e. after food intake) blood glucose are undesired from a health point of view, as they may promote development of diabetes type II in the individual and/or for other reasons like high insulin response. Thus, there is a desire to reduce post-prandial blood glucose levels, in particular when such is connected to consuming foods rich in high-glycaemic carbohydrates and/or sugars. The Institute of Medicine's Food and Nutrition Board (IOM/FNB, 1994) defined functional foods as "any food or food ingredient that may provide a health benefit beyond the traditional nutrients it contains". Thus, functional foods may deliver a health benefit to the consumer beyond that of calories, vitamins, minerals and other nutrients. There is an increasing demand for functional foods, e.g. for functional foods that can suppress or ameliorate post-prandial blood glucose, as mentioned above.

The compound 1 -deoxynojirimycin (generally abbreviated as "DNJ") is a potent o glucosidase inhibitor, and as such may suppress postprandial blood glucose concentrations. DNJ is present in e.g. various parts of the mulberry plant, such as leaves, bark and fruit. At present, teas containing mulberry are commercially supplied as functional foods in many countries. Such teas are (e.g. from a taste-perspective) not preferred by all consumers, and for efficacy would need to be consumed within a fairly short time frame of consuming carbohydrate-rich foods. Thus, it would be more convenient if a DNJ-rich food can be provided that can contain and supply the right dose of DNJ to the person consuming such, and which food can easily be combined with carbohydrate-rich foods, such as e.g. bread or cookies.

Edible fat continuous emulsions are well known in the art and include for example spreads, margarines, and (ingredients for use in) confectionary products. Spreads are usually intended and used for spreading on bread (slices or rolls), whereas margarines often are considered the harder products sold e.g. in wrappers and which are e.g. used for making baked goods such as cookies and cakes. These fat-continuous emulsions like spreads and margarines could be a good carrier of DNJ (e.g. from an extract of mulberry such as mulberry fruit), as they are combined often with carbohydrate-rich foods. Spread is rarely consumed on its own, but generally used for spreading on bread, and margarines are e.g. used for baking cakes or cookies. Thus, these emulsions would be an excellent carrier for DNJ and DNJ-containing extracts of mulberry (leaf, bark, fruit) in terms of being consumed generally with carbohydrate-rich foods, and often the ratio emulsion : carbohydrate consumed is fairly predictable, and thus the DNJ content on the emulsion can be tailored fairly well so that an optimal ratio DNJ : carbohydrate is consumed. Apart from being able to be consumed easily in a desired ratio with carbohydrate-rich foods such as bread, cakes and cookies, the DNJ-containing food should also be able to release the DNJ in the stomach quickly, as it is believed that a slow release may render the DNJ not fully effective: the alpha-glucosidase inhibitory effect should be available in the stomach quickly after ingestion of the carbohydrate-rich food, i.e. in a similar time frame of the breakdown of starch to glucose.

It was found that on this point many fat-continuous emulsions do not perform satisfactorily. In short: it was found that DNJ-release from a simple fat-continuous emulsion spread on a slice of bread in a model stomach was not quick enough, e.g. when compared to the situation when DNJ was sprinkled on top of the same slice of bread spread with a similar fat-continuous emulsion (i.e. the DNJ not being present in the emulsion but separately added on top). The DNJ in this case was used in the form of a spray-dried aqueous extract of white mulberry fruit. Thus, one can say that from a DNJ-release point of view fat-continuous emulsions such as spreads and margarines are not a very suitable vehicle for DNJ, and that rather DNJ is separately added just before consumption. Still, incorporation of DNJ (e.g. in the form of an aqueous extract of white mulberry fruit) into the fat-continuous emulsion is desired for reasons of convenience and correct dosing.

Thus, there is a need for an edible fat-continuous emulsion, such as e.g. a spread or margarine, containing DNJ, in particular in the form of an (aqueous) extract of mulberry white fruit, which shows good release of the DNJ when such emulsion is spread on a slice of bread and such slice of bread with DNJ-containing spread is tested in a model of a stomach. Preferably, the release of the DNJ should be more close to what can be achieved when the DNJ (in the form of an aqueous mulberry fruit extract) is sprinkled on top of a slice of bread spread with the same emulsion without DNJ. Preferably, such DNJ-release should not only be good in a model stomach, but also in a human, but such is more difficult to measure than a model stomach.

Summary of the Invention

It has now been found that the objectives as set out above may be achieved, at least in part, by an edible fat-continuous emulsion, which emulsion comprises from 20 to 85% by weight of a continuous fat phase, from 15 to 80% by weight of a dispersed water phase, the amount of water and fat and oil together being at least 75% by weight, wherein the emulsion product further comprises from 0.001 to 0.08 % by weight of 1 - deoxynojirimycin (DNJ) and from 0.05 to 1 % by weight of lecithin, wherein the lecithin comprises phosphatidylcholine (PC), phosphatidylinositol (PI), and

phosphatidylethanolamine (PE) wherein the weight ratio (PC+PI)/PE in the emulsion is at least 2.5.

The invention also relates to a process for preparing such emulsions, which process comprises the steps of preparing a aqueous phase containing the desired amount of 1- deoxynojirimycin, preparing an oil phase containing the desired amount of lecithin, wherein the lecithin comprises phosphatidylcholine (PC), phosphatidylinositol (PI), and phosphatidylethanolamine (PE) wherein the weight ratio (PC+PI)/PE in the emulsion is at least 2.5, and mixing and emulsifying the aqueous phase and oil phase so that a fat- continuous emulsion is obtained.

The invention further relates to the use of the emulsion according to this invention, wherein the emulsion is combined with edible matter comprising carbohydrates, in such an amount that the resulting foodstuff contains from 0.001 to 0.03 % by weight of DNJ on carbohydrate basis.

Detailed description of the invention

The compound 1 -deoxynojirimycin is commonly abbreviated in literature as "DNJ". The lUPAC name for it is: (2R,3R,4R,5S)-2-(hydroxymethyl)piperidine-3,4,5-triol. The compound is an alpha-glucosidase inhibitor, and is commonly found in parts of the mulberry plant (e.g. leaves, bark, fruit). For measuring amounts and concentrations of DNJ: the method of the examples herein is to be used. DNJ is herein to be understood as to comprise both DNJ and the edible salts thereof. The measurement method measures the sum of these.

"Lecithin" is herein to be understood as commercial lecithin as used by food

manufacturers and as is allowed for use in foods as E322. Lecithin can be obtained by water degumming the extracted oil of seeds. It is a mixture of various phospholipids and other components, and the composition depends on the origin of the lecithin. A major source of lecithin is soybean oil. Most relevant phospholipids found in lecithin include phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, and phosphatidic acid.

The words "fat" and "oil" are used interchangeably herein, and refer to edible triacylglycerides of fatty acids. The person of average skill in the art will know that for such emulsion products to be spreadable (e.g. as a "spread") and in order to satisfy criteria such as health, mouthfeel and appearance, at least part of the continuous (lipid) phase (the fat and oil together) needs to be solid at room temperature. Still, for health reasons part of the continuous phase is preferably oil.

Weight percentages are herein to be understood as to be based on weight % based on the total emulsion, unless specified differently.

In the emulsion according to the present invention, it is preferred (e.g. for reasons of product properties) that the emulsion comprises from 35 to 80%, more preferably from 40 to 80%, by weight on the emulsion of a continuous fat phase. The emulsions according to the present invention are preferably spreads or

margarines. The fat phase of such fat-continuous emulsions like spreads and margarines are preferably a mixture of liquid oil (i.e. fat that is liquid at ambient temperature) and fat which is solid at ambient temperatures. The solid fat, also called structuring fat or hardstock fat, serves to structure the fat phase and helps to stabilize the aqueous phase by forming a fat crystal network. Thus (and also for health reasons), in the present invention, preferably 20 to 90 % by weight (more preferably 40 to 90%) of the continuous fat phase is oil, and 10 to 80% by weight (more preferably 10 to 60%) is preferably a hardstock fat. A preferred oil in this connection is vegetable oil, such as sunflower oil, rapeseed oil, linseed oil, or soy bean oil.

For reasons of efficacy, the emulsion of the present invention preferably comprises from 0.002 to 0.05 % by weight, more preferably from 0.003 to 0.04 % by weight, of 1- deoxynojirimycin (DNJ), on the emulsion. For reasons of emulsion stability, mouthfeel, and efficacy in DNJ release in the stomach the emulsion of the present invention preferably comprises from 0.08 to 0.6 % by weight of lecithin, more preferably from 0.1 to 0.4%. It was found by the present inventors that in terms of speed of release of DNJ in the stomach, as determined by the stomach model as used herein, not each and every lecithin behaves the same. In lecithin the proportions of the various phospholipids vary, e.g. depending on origin and method of production, isolation, and purification. It was found that for speedy release of the DNJ, yet not affecting properties like emulsion stability and mouthfeel not too negatively, it is required that the lecithin in the present emulsion comprises phosphatidylcholine (PC), phosphatidylinositol (PI), and phosphatidylethanolamine (PE) wherein the weight ratio (PC+PI)/PE in the emulsion is at least 2.5. More preferably, for such reasons of efficacy and product properties, said weight ratio (PC+PI)/PE is at least 3.0.

As stated, the emulsions according to the present invention are preferably spreads or margarines, as such spreads can be used for spreading on bread, and thus

carbohydrate from bread is consumed with DNJ to regulate blood sugar levels after intake of such spread-covered slice of bread. The margarines according to the above can be used conveniently in making baked goods such as cookies and cakes. Such cake or cookie will have the advantage of containing DNJ, and thus eating such cake or cookie is expected to lead to a lower post prandial blood glucose level compared when an ordinary margarine (not containing DNJ) is used for preparing such cake or cookie.

The spreads and margarines according to the present invention preferably will have a Stevens value of between 50 and 350.

The Stevens value of a W/O emulsion according to the invention is determined as follows. A product, whereof the Stevens-value is to be measured is stabilized at 5 degrees Celsius. The hardness of the product is measured with a Stevens

penetrometer (Brookfield LFRA Texture Analyser (LFRA 1500), ex Brookfield

Engineering Labs, UK) equipped with a stainless steel probe with a diameter of 6.35 mm and operated in "normal" mode (the container used for the spreads was a 250g tub as commercially used for spreads). The probe is pushed into the product at a speed of 2 mm/s, a trigger force of 5 gram from a distance of 10 mm. The force required is read from the digital display and is expressed in grams. Preferably the emulsion according to the invention has a Stevens-value below 300, more preferably below 250, even more preferably below 220, most preferably below 200. It is preferred that the Stevens value of the spreads according to the present invention have a Stevens value of from 80 to 250, more preferably from 90 to 220, even more preferably 100 to 200 and still even more preferably of from 120 to 200. The values in this are measured after cycling. In the present invention, the DNJ is preferably incorporated into the emulsion according to the invention as an extract of part of a plant of the Mulberry family (the Morus sp tree), more preferably an extract of the fruit of mulberry. Plants of the Mulberry family are known to contain DNJ. Even more preferably, the extracts as described above are obtained by aqueous extraction, which may be dried to provide a powder. Thus, it is preferred that the emulsion according to the present invention comprises from 0.5 to 10% of extract of mulberry, preferably from mulberry fruit, more preferably from fruits from the white mulberry tree (Morus alba).

Mulberry fruit extract can be obtained from the fruit of the Morus sp tree. Morus sp is widely cultivated in Asia, especially in China, where the leaves are used to feed silkworms employed in the commercial production of silk. There are white mulberry varieties (such as Morus alba L) which fruit remains white upon ripening, while other varieties (such as Morus nigra L or Morus rubra L) the fruit turns pink to dark red upon ripening. The ripe mulberry fruits are widely used in juice, beverages, wine, jams, pies and tarts in many countries, such as China, Japan and India. Dried mulberry fruit has been used as a traditional Chinese medicine for more than 2000 years, and its uses are shown in the Chinese Pharmacopeia. Mulberry fruit is also mentioned in the Ayuverdic Materia Medica. Depending on the taste preference, the emulsion according to the present invention preferably comprises 0.1-1.5% by weight of edible salt (i.e. NaCI and/or KCI).

Depending on e.g. the desired fat level the formulations according to the present invention may further comprise emulsifiers. Hence, the emulsion product according to the present invention may further comprise a mono acyl glyceride emulsifier. An ingredient that also may be present is sorbate, as a preservative.

Preferably the emulsion according to the invention has water droplet size with a D3,3 below 12, more preferably from 12 to 3, even more preferably from 8 to 3, yet even more preferably from to 6 to 3.2 and most preferably from 4.5 to 3.5. Preferably the emulsion according to the invention has water droplet size distribution with an e-sigma of at most 3, more preferably at most 2.5, yet more preferably of at most 2.3, even more preferably of at most 1.8 and most preferably of at most 1 .4.

Water droplet size and distribution is herein measured by the following method.

The normal terminology for Nuclear Magnetic Resonance (NMR) is used throughout this method. On the basis of this method the parameters D3,3 and exp(o) of a lognormal water droplet size distribution can be determined. The D3,3 is the volume weighted mean droplet diameter and σ (e-sigma) is the standard deviation of the logarithm of the droplet diameter. The NMR signal (echo height) of the protons of the water in a water -in-oil emulsion are measured using a sequence of 4 radio frequency pulses in the presence (echo height E) and absence (echo height E ^* ) of two magnetic field gradient pulses as a function of the gradient power. The oil protons are suppressed in the first part of the sequence by a relaxation filter. The ratio (R=E/E ^* ) reflects the extent of restriction of the translational mobility of the water molecules in the water droplets and thereby is a measure of the water droplet size. By a

mathematical procedure -which uses the log-normal droplet size distribution - the parameters of the water droplet size distribution D3,3 (volume weighed geometric mean diameter) and σ (distribution width) are calculated. A Bruker magnet with a field of 0.47 Tesla (20 MHz proton frequency) with an air gap of 25 mm is used (NMR Spectrometer Bruker Minispec MQ20 Grad, ex Bruker Optik GmbH, DE). The droplet size of the spread is measured, according to the above described procedure, of a spread stabilized at 5 degrees Celsius right after production for one week. This gives the D3,3 after stabilization at 5 degrees Celsius.

Preferably, the products according to the present invention have no free water (as measured with Wator test-paper). EXAMPLES Sample design

Three ways of combining spray-dried mulberry fruit extract (from aqueous extraction) (MFE, to provide DNJ) with a carbohydrate source were tested on liberation of DNJ:

A. MFE formulated in water phase of water-in-oil emulsion (MFE-containing 39% fat spread) and then spread on bread

B. MFE powder sprinkled on bread covered with 39% fat spread

C. MFE powder sprinkled on cooked Sona Masuri rice

D. As sample A, but now the emulsion contained 0.25% of a specific lecithin

Materials and Methods

Materials

• White bread

• White, cooked Sona Masuri rice

• 0.50g MFE/20g spread (W/O, 39% fat)

• Reference W/O spread, 39% fat

• Freeze-dried Mulberry fruit extract (see methods below)

• USP (United States Pharmacopeia) Apparatus 2 dissolution equipment: USP <71 1 >

• Gastric juice (see below) Chemicals

· Amano lipase A from Aspergillus niger - Sigma 534781

• Pepsin from porcine gastric mucosa- Sigma P7125

• Sodium chloride from - Sigma S9888

• Sodium taurocholate from -Sigma T4009

Mulberry fruit extract (MFE) preparation

Purification of the dried extract of white mulberry fruit Mulberry fruit extract as a spray-dried powder was supplied by Draco Natural Products, USA.

Of the above MFE powder, 4.1 % was dissolved in demi water at 40± 5 °C and stirred continuously for 1 hour using a "paint" mixer. After removing insoluble MFE ingredients by centrifugation in centrifuge : Sigma 8K from Sigma, 1.5 liters bucket in swing-out rotor, max 5100 rpm (=8578 xg), for 45 minutes. The pellet (insoluble material) was discarded, and the supernatant was collected and pooled, then immediately freeze dried, for storage.

Three batches so-prepared were mixed, in order to ensure uniformity and consistency. Analysis has shown that the DN J-content of this was 0.48%.

Gastric juice composition

Table 1 : Gastric juice composition

Fill up to 500 ml with deionized water and adjust pH to 1.6 with HCI.

Equipment

Rice cooker Remo Item 89405

USP (United States Pharmacopeia) dissolution equipment, model 2, Vankel VK 7000, schematically as in figure 1 as in-vitro model for the stomach.

In-vitro model

The full model is based on three consecutive steps

1. Oral cavity

2. Gastric step

3. Small intestine step

In this example, only the gastric step is performed, and intestinal step is not included. Gastric digestion conditions

Digestive enzymes are pepsin and li

Digestion temperature was 37 °C

Digestion time of 2 hours

Operating speed 100 rpm, pH 1.6

Gastric dissolution

Preparation of the 3 different matrices

A. 20 g of 39% fat spread at 2.5% MFE (i.e. absolute amount of 500 mg MFE) was spread on 3 slices of white bread (50 g Carbohydrates). After that, the spreaded bread was cut into small pieces of 2-3 cm.

B. 20 g of 39% fat reference spread (i.e. without MFE) was spread on 3 slices of white bread (50 g Carbohydrates). After that, 500mg MFE powder was sprinkled on top of the spread. Then the sandwich was slightly pressed and strips were cut crosswise into 2-3 cm cubes.

C. 62 g of Sona Masuri rice (50g Carbohydrates) was cooked in rice cooker, and then 500 mg of MFE was sprinkled on top and mixed with a spoon.

D. As sample A, but now the spread further contained 0.25% lecithin (with a ratio (PC+PI)/PE of about 4.4), and slightly less water

Spread composition

A set of 39% fat spreads with and without MFE, and for a spread with MFE with and without lecithin, has been processed on pilot plant scale under food-grade conditions and two tubs each were available for in vitro evaluation. Table 2 sets out the composition of the spread A (with MFE) tested. The spread of example B had the same overall composition as A, except that no mulberry fruit extract was included in the aqueous phase. The spread of example D had the same overall composition as A, except that it further contained a lecithin (which lecithin comprises phosphatidylcholine (PC), phosphatidylinositol (PI), and phosphatidylethanolamine (PE) wherein the weight ratio (PC+PI)/PE in the emulsion was about 4.4). Table 2: spread composition

The properties of the spread of A and B are in table 3.

Table 3: properties of the 39% fat spreads, stored at 5°C

Spread of Spread of Spread of

Aqueous phase sample B sample A sample D

Directly after processing

Water droplet size (D3.3) [μηι] 8.6 9.0 5.6

E ^A sigma 1.66 1 .71 1 .6

Stevens hardness (St.6.35) [g] 24 16 15

Free water (WATOR) 0 0

Moisture content [%] 60.8 58.5

After 1 day storage at 5°C

Water droplet size (D3.3) [ m] 8.7 9.2

E ^A sigma 1.71 1 .74

Stevens hardness (St.6.35) [g] 45 44 Free water (WATOR) 0 0

After 1 week storage at 5°C

Water droplet size (D3.3) [μηι] 8.8 9.3 8.8

E ^A sigma 1.75 1 .74 1 .75

Stevens hardness (St6.3s) [g] 70 74 86.7

Free water (WATOR) 0 0

After 5 weeks storage at

5°C

Water droplet size (D3.3) [μηι] 6.0

E ^A sigma 1 .7

Stevens hardness (St6.3s) [g] 109

After 10 weeks storage at

5°C

Water droplet size (D3.3) [Mm] n.d. 9.5

E ^A sigma n.d. 1 .78

Stevens hardness (St6.35) [g] n.d. 107

After 16 weeks storage at

5°C

Water droplet size (D3.3) [Mm] n.d. 10.1

E ^A sigma n.d. 1 .65

Stevens hardness (St6.35) [g] n.d. 56

Free water (WATOR) n.d. 0

Preparation of cooked rice

Sona Masoori

Rice weight [g] 62.01

Pan empty [g] 146.68

Cook water [ml] 290.06

Cooking time [m:s] 23:13

Keep warm time [m] 10 Gastric digestion process

For simulating the gastric step, the USP dissolution apparatus was used. The temperature of the water was set up to 37°C. The slow shear in the stomach was simulated with a special paddle at a stirring rate of 100 rpm.

700 ml deionised water was added in 1 I USP flask and introduced in the dissolution model. After the temperature had reached 37°C, 50 ml of gastric juice was added and immediately the bread cubes or rice was added, to start the simulated gastric digestion. 2ml samples were removed at different time of digestion, and immediately centrifuged at 12000 rpm for 10 min.

Supernatant was analyzed by LC-MS method (see below).

Quantitative DNJ analysis

DNJ was quantified in the water phase of gastric digestion of USP by a quantitative LC- MS method (see below).

LC-MS analysis

Quantitative analysis was carried out on an Agilent HPLC system equipped with a triple quadrupole mass spectrometer (6410 Agilent, Amstelveen, the Netherlands).

The column used in all experiments was a TSK gel Amide-80 HPLC column of 2.0 mm inner diameter and a length of 150 mm, packed with 5 μηη particles (TOSOH, Japan). Injection volume was 5 μί. Column temperature was 30 ^° C. A mobile phase consisting of 0.1 % formic acid in acetonitrile (A) and 0.1 % formic acid in Milli-Q water (B) and a flow rate of 0.2 ml/min was used in all experiments.

Multiple reaction monitoring (MRM) transitions were used for quantification. 1- Deoxynojirimycin (purity > 95%, Sigma) was used for the quantification and Miglitol (purity > 98%, Sigma) was used as an internal standard. Calibration curves in the range 0.15 - 13 μg g were made.

Multiple reaction monitoring (MRM) transitions were used for Miglitol (I.S.)

quantification.

LC-MS system Binair high pressure 1200 pump (Agilent, Amstelveen, the Netherlands

Quadrupole tandem MS system (6410 Agilent, Amstelveen, the Netherlands)

Chromatographic conditions

Analytical column: Gel Amide-80 150x2.0 mm, 5 μηι, (TSK - Tosoh)

Mobile phase A: acetonitrile + 0.1 % formic acid

Mobile phase B: Milli-Q water + 0.1 % formic acid

Flow rate: 0.2 ml/min

Column temperature: 30°C

Injection volume: 5 μΙ

Run time: 15 min.

Retention time DNJ: 10.5 min

Results

From the above, it became clear that based on in-vitro data, mimicking gastric digestion, DNJ release in the gastric phase depends on emulsion type, food matrix, and (for an emulsion) the presence of a certain lecithin. The results are graphically displayed in figure 2.